Rotator cuff tears of the shoulder result in annual economic burden of $3-4 billion in surgical expenses alone and have major impact on individual quality of life. Suture repair is the standard of care but high re- tear rates have been reported. Extracellular matrix (ECM) augmentation scaffolds are used to try and improve outcomes for massive tears because they contain bioactive molecules that stimulate cell migration, proliferation and ECM synthesis. However, there are limitations to all the currently available scaffolds and they do not recreate the native tendon-to-bone interface, a graded tendon-fibrocartilage-bone composite tissue which functions to reduce stress concentration at the tendon insertion. Therefore there is a need for a device that more closely recapitulates structure and function of the native tendon-bone interface. Our overall goal is to develop a biomaterial scaffold that promotes integrated tendon-to-bone formation for use in rotator cuff repair. To achieve this goal, we will take an innovative approach to challenge the current paradigm for scaffold development. In addition to ECM cues, other microenvironmental factors such as scaffold microarchitecture can control stem cell differentiation and the formation of complex tissues. For example, electrospinning can be used to form fibers with controllable fiber alignment patterns and diameters, each of which induces specific differentiation responses by stem cells. The current paradigm of scaffold development involves selecting several candidate scaffolds, examining cell behavior in response to culture on these scaffolds, and then modifying scaffold design based on the results obtained before repeating the process. With this approach, there is limited ability to independently manipulate and integrate microenvironmental variables (e.g. scaffold fiber diameter and anisotropy, cell-adhesive ECM ligands) that critically affect cel differentiation and the functional properties of resulting tissue. Therefore, our understanding of how scaffold microenvironments affect functional outcomes remains limited, and identifying scaffold conditions that promote functional composite tissue formation is a highly inefficient process. To overcome these limitations, we intend to use an in vitro micro-photopatterning (microPP) technique to systematically screen scaffold fiber diameter and anisotropy for desired effects on stem cell differentiation towards tendon, cartilage and bone. We will then further functionalize the microPP architectures with tendon-, cartilage- and bone-specific ECM to evaluate additional benefit conferred with the integration of ECM specific ligands. Finally, we wil apply these findings to a multi-layered electrospun scaffold that will be evaluated in vitro for it ability to promote development of a vertically graded tendon- fibrocartilage-bone interface, similar to that seen at the normal tendon-bone interface of the rotator cuff. These findings will improve understanding of microenvironmental cues for tendon-bone tissue engineering and are expected to improve tissue engineered regeneration of the rotator cuff tendon-bone interface.